A single nucleotide polymorphism in the human serotonin transporter introduces a new site for N-linked glycosylation

Trine Nygaard Rasmussen; Per Plenge; Tina Bay; Jan Egebjerg; Ulrik Gether

doi:10.1016/j.neuropharm.2009.05.009

A single nucleotide polymorphism in the human serotonin transporter introduces a new site for N-linked glycosylation

Trine Nygaard Rasmussen, Per Plenge, Tina Bay, Jan Egebjerg, Ulrik Gether

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

21 Citationer (Scopus)

Abstract

Udgivelsesdato: 2009-Sep

Originalsprog	Engelsk
Tidsskrift	Neuropharmacology
Vol/bind	57
Udgave nummer	3
Sider (fra-til)	287-94
Antal sider	7
ISSN	0028-3908
DOI	https://doi.org/10.1016/j.neuropharm.2009.05.009
Status	Udgivet - 2009

Bibliografisk note

Keywords: Amino Acid Sequence; Animals; Binding Sites; Cell Line; Cells, Cultured; Cerebral Cortex; Citalopram; Gene Expression; Glycosylation; Humans; Kinetics; Molecular Sequence Data; Neurons; Polymorphism, Single Nucleotide; Rats; Rats, Wistar; Serotonin; Serotonin Plasma Membrane Transport Proteins; Serotonin Uptake Inhibitors

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10.1016/j.neuropharm.2009.05.009

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Citationsformater

@article{4590c390698b11df928f000ea68e967b,

title = "A single nucleotide polymorphism in the human serotonin transporter introduces a new site for N-linked glycosylation",

abstract = "The human serotonin transporter (hSERT) is responsible for reuptake of serotonin (5-HT) from the synaptic cleft and is target for antidepressant medicine. Differential hSERT activity caused by genetic polymorphisms is believed to affect the risk of developing depression and, moreover, to affect the response to antidepressant therapy. The hSERT contains in the second extracellular loop (EL2) two sites for N-linked glycosylation that are critical for functional transporter expression. Here we examine a non-synonymous single nucleotide polymorphism (SNP) in EL2 that gives rise to a potential third glycosylation site due to substitution of a lysine at position 201 with an asparagine (K201N). In agreement with introduction of an additional glycosylation site, western blot analysis showed migration of hSERT K201N corresponding to a higher molecular weight than wild type hSERT upon expression in both HEK293 cells and primary cultures of cortical neurons. An increase in molecular weight was not observed after removal of glycans with peptide N-glycosidase F (PNGase F). Quantitative analysis of western blots indicated significantly increased total transporter expression ( approximately 30%) for hSERT K201N as compared to hSERT in both cell systems. The increase in expression was accompanied by corresponding significant increases in the number of [(3)H]citalopram binding sites and in the V(max) for [(3)H]5-HT uptake. Characterization of mutants carrying all possible combinations of glycosylation sites demonstrated clear correlation between the number of glycosylation sites and the level of transporter activity, and showed that K201N could substitute for either one of the two original glycosylation sites.",

author = "Rasmussen, {Trine Nygaard} and Per Plenge and Tina Bay and Jan Egebjerg and Ulrik Gether",

note = "Keywords: Amino Acid Sequence; Animals; Binding Sites; Cell Line; Cells, Cultured; Cerebral Cortex; Citalopram; Gene Expression; Glycosylation; Humans; Kinetics; Molecular Sequence Data; Neurons; Polymorphism, Single Nucleotide; Rats; Rats, Wistar; Serotonin; Serotonin Plasma Membrane Transport Proteins; Serotonin Uptake Inhibitors",

year = "2009",

doi = "10.1016/j.neuropharm.2009.05.009",

language = "English",

volume = "57",

pages = "287--94",

journal = "Neuropharmacology",

issn = "0028-3908",

publisher = "Pergamon Press",

number = "3",

}

TY - JOUR

T1 - A single nucleotide polymorphism in the human serotonin transporter introduces a new site for N-linked glycosylation

AU - Rasmussen, Trine Nygaard

AU - Plenge, Per

AU - Bay, Tina

AU - Egebjerg, Jan

AU - Gether, Ulrik

N1 - Keywords: Amino Acid Sequence; Animals; Binding Sites; Cell Line; Cells, Cultured; Cerebral Cortex; Citalopram; Gene Expression; Glycosylation; Humans; Kinetics; Molecular Sequence Data; Neurons; Polymorphism, Single Nucleotide; Rats; Rats, Wistar; Serotonin; Serotonin Plasma Membrane Transport Proteins; Serotonin Uptake Inhibitors

PY - 2009

Y1 - 2009

N2 - The human serotonin transporter (hSERT) is responsible for reuptake of serotonin (5-HT) from the synaptic cleft and is target for antidepressant medicine. Differential hSERT activity caused by genetic polymorphisms is believed to affect the risk of developing depression and, moreover, to affect the response to antidepressant therapy. The hSERT contains in the second extracellular loop (EL2) two sites for N-linked glycosylation that are critical for functional transporter expression. Here we examine a non-synonymous single nucleotide polymorphism (SNP) in EL2 that gives rise to a potential third glycosylation site due to substitution of a lysine at position 201 with an asparagine (K201N). In agreement with introduction of an additional glycosylation site, western blot analysis showed migration of hSERT K201N corresponding to a higher molecular weight than wild type hSERT upon expression in both HEK293 cells and primary cultures of cortical neurons. An increase in molecular weight was not observed after removal of glycans with peptide N-glycosidase F (PNGase F). Quantitative analysis of western blots indicated significantly increased total transporter expression ( approximately 30%) for hSERT K201N as compared to hSERT in both cell systems. The increase in expression was accompanied by corresponding significant increases in the number of [(3)H]citalopram binding sites and in the V(max) for [(3)H]5-HT uptake. Characterization of mutants carrying all possible combinations of glycosylation sites demonstrated clear correlation between the number of glycosylation sites and the level of transporter activity, and showed that K201N could substitute for either one of the two original glycosylation sites.

AB - The human serotonin transporter (hSERT) is responsible for reuptake of serotonin (5-HT) from the synaptic cleft and is target for antidepressant medicine. Differential hSERT activity caused by genetic polymorphisms is believed to affect the risk of developing depression and, moreover, to affect the response to antidepressant therapy. The hSERT contains in the second extracellular loop (EL2) two sites for N-linked glycosylation that are critical for functional transporter expression. Here we examine a non-synonymous single nucleotide polymorphism (SNP) in EL2 that gives rise to a potential third glycosylation site due to substitution of a lysine at position 201 with an asparagine (K201N). In agreement with introduction of an additional glycosylation site, western blot analysis showed migration of hSERT K201N corresponding to a higher molecular weight than wild type hSERT upon expression in both HEK293 cells and primary cultures of cortical neurons. An increase in molecular weight was not observed after removal of glycans with peptide N-glycosidase F (PNGase F). Quantitative analysis of western blots indicated significantly increased total transporter expression ( approximately 30%) for hSERT K201N as compared to hSERT in both cell systems. The increase in expression was accompanied by corresponding significant increases in the number of [(3)H]citalopram binding sites and in the V(max) for [(3)H]5-HT uptake. Characterization of mutants carrying all possible combinations of glycosylation sites demonstrated clear correlation between the number of glycosylation sites and the level of transporter activity, and showed that K201N could substitute for either one of the two original glycosylation sites.

U2 - 10.1016/j.neuropharm.2009.05.009

DO - 10.1016/j.neuropharm.2009.05.009

M3 - Journal article

C2 - 19500602

SN - 0028-3908

VL - 57

SP - 287

EP - 294

JO - Neuropharmacology

JF - Neuropharmacology

IS - 3

ER -